In order to test the granule exocytosis model for lymphocyte cytotoxicity, we have examined the cytotoxic activity of RBL cells transfected with genes for cytotoxic lymphocyte granule components. RBL, a rat mucosal mast cell tumor line which degranulates in response to cross-linking its IgE Fc receptor is not cytotoxic but acquires a potent lytic activity against IgE coated red cells when transfected with the mouse cytolysin (cy) gene. This potency is equivalent to that of cloned CTL with this target. We have found that electroporation gives much higher (2 orders of magnitude) transfection efficiency than the calcium phosphate technique originally used. Furthermore the cloned RBL electroporation transfectants (RBL-cy) give even more potent lytic activity against RBC targets than did those generated with calcium phosphate. Using tumor target cells, the RBL-cy give good cytotoxicity, although not as potent as cloned CTL. When target DNA degradation was examined with RBL-cy effector cells, it was not detectable, in striking contrast to that induced by CTL in the same experiment. Thus the cell-- delivered cytolysin mimics the effects of purified cytolysin added to the medium. We have constructed double and single RBL transfectants expressing cytolysin and the granule serine protease granzyme A (gza). As expected, RBL-gza transfectants with protein expression levels comparable to cloned CTL show no cytotoxic activity. RBL-cy-gza transfectants showing good expression of both these granule components showed cytolytic activity comparable to RBL-cy on both RBC and tumor targets. When DNA breakdown in tumor targets was examined, it was clearly observed with RBL-cygza effectors, although not as potently as with CTL effectors. These preliminary results support our earlier evidence suggesting that granzyme A triggers target DNA breakdown after gaining access to the target cell cytoplasm.